Previously we have shown that extracellular (ex) heat shock protein (HSP)60 causes cardiac myocyte apoptosis and the synthesis of TNF and IL-1$. Blocking toll-like receptor (TLR)4 decreased, but did not abolish apoptosis, suggesting that the interaction of HSP60 with TLR4 is complex and involves other proteins. Our over-arching hypothesis is that HSP60 can cause apoptosis and inflammation, and this is associated with abnormal expression, distribution or post-translational modification of HSP60. We will expand our investigation in this important area with 3 specific aims designed to further define the role of HSP60 in heart failure and other cardiovascular disease. SA1 - Investigate the mechanism(s) of exHSP60 mediated apoptosis and inflammation. We will extend our investigation of TLR4 in exHSP60 signaling, determine whether, Eritoran, an inhibitor of TLR4, can reduce apoptosis in vitro and in vivo, and further characterize plasma membrane associated HSP60. SA2 - Investigate extracellular HSP60 trafficking and the role of post-translational modification in its toxicity. We hypothesize that post-translational modifications of HSP60 influence its localization and its extracellular toxicity. Studies have found high levels of HSP60 in the blood of patients with diabetes and other diseases. We will investigate the role of post- translational modifications as well as exosomes in the toxicity of this HSP60. Second we will examine the source of membrane associated HSP60. SA3 - Investigate the role of HSP60 in Repetitive Injury and the Heat Shock Paradox - The heat shock response is protective, and yet, when the heat shock response is induced after an inflammatory stress, rather than protection, increased injury occurs, hence. We found that HSP60 is increased after inflammation followed by heat shock, but not when the order of these stimuli is reversed. This increase in HSP60 is associated with increased apoptosis. In addition, blocking activated heat shock factor (HSF) decreased apoptosis. We will investigate the role of HSF1 and 2 in the heat shock paradox with selective knockout models. HSP60's role in the heat shock paradox will be studied by examining its cellular localization and manipulating its expression. The planned work will expand the PI's investigation of HSP60 and its role in cardiovascular inflammation and apoptosis and has the potential to lead to new therapeutic approaches to heart failure.